RESEARCH PAPER

Nitric oxide donor trans-[RuCl([15]aneN4)NO]2+ as apossible therapeutic approach for Chagas’ diseasebph_576 1..13

Paulo MM Guedes1, Fabiana S Oliveira2, Fredy RS Gutierrez1, Grace Kelly da Silva1,Gerson Jhonatan Rodrigues3, Lusiane Maria Bendhack3, Douglas W Franco4,Maria A Do Valle Matta5, Dario S Zamboni6, Roberto Santana da Silva2 and João Santana Silva1

1Department of Biochemistry and Immunology, School of Medicine at Ribeirão Preto, São Paulo, Brazil, 2Department ofChemistry, School of Pharmaceutical Sciences at Ribeirão Preto, São Paulo, Brazil, 3Department of Pharmacology, School ofMedicine at Ribeirão Preto, São Paulo, Brazil, 4Institute of Chemistry at São Carlos, São Paulo, Brazil, 5Laboratory of CellularUltrastructure, Oswaldo Cruz Institute-FIOCRUZ, Rio de Janeiro, Brazil, and 6Department of Cell Biology, School of Medicineat Ribeirão Preto, University of São Paulo, São Paulo, Brazil

Background and purpose: Benznidazole (Bz) is the therapy currently available for clinical treatment of Chagas’ disease.However, many strains of Trypanosoma cruzi parasites are naturally resistant. Nitric oxide (NO) produced by activatedmacrophages is crucial to the intracellular killing of parasites. Here, we investigate the in vitro and in vivo activities againstT. cruzi, of the NO donor, trans-[RuCl([15]aneN4)NO]2+.Experimental approach: Trans-[RuCl([15]aneN4)NO]2+ was incubated with a partially drug-resistant T. cruzi Y strain and theanti-proliferative (epimastigote form) and trypanocidal activities (trypomastigote and amastigote) evaluated. Mice were treatedduring the acute phase of Chagas’ disease. The anti-T. cruzi activity was evaluated by parasitaemia, survival rate, cardiacparasitism, myocarditis and the curative rate.Key results: Trans-[RuCl([15]aneN4)NO]2+ was 10- and 100-fold more active than Bz against amastigotes and trypomastigotesrespectively. Further, trans-[RuCl([15]aneN4)NO]2+ (0.1 mM) induced 100% of trypanocidal activity (trypomastigotes forms)in vitro. Trans-[RuCl([15]aneN4)NO]2+ induced permanent suppression of parasitaemia and 100% survival in a murine model ofacute Chagas’ disease. When the drugs were given alone, parasitological cures were confirmed in only 30 and 40% of theanimals treated with the NO donor (3.33 mmol·kg-1·day-1) and Bz (385 mmol·kg-1·day-1), respectively, but when giventogether, 80% of the animals were parasitologically cured. The cured animals showed an absence of myocarditis and anormalisation of cytokine production in the sera. In addition, no in vitro toxicity was observed at the tested doses.Conclusions and implications: These findings indicate that trans-[RuCl([15]aneN4)NO]2+ is a promising lead compound forthe treatment of human Chagas’ disease.British Journal of Pharmacology (2010) doi:10.1111/j.1476-5381.2009.00576.x

Keywords: NO donors; trans-[RuCl([15]aneN4)NO]2+; benznidazole; Trypanosoma cruzi; Chagas’ disease

Abbreviations: 4-pic, 4-picoline; AA, ascorbic acid; ABTS, (2,2′-azino-di-(3-ethyl benzthiazoline-6-sulphonic acid); BMDM,bone marrow-derived macrophages; BT, bloodstream trypomastigote forms of Trypanosoma cruzi; Bz, ben-znidazole; DAF-2 DA, 4,5-diaminofluorescein diacetate; DAF-2T, DAF-2 triazole; DMEM, Dulbecco’s modifiedEagle’s medium; FBS, fetal bovine serum; FI, fluorescence intensity; FITC, fluorescein isothiocyanate; IFN-g,interferon-g; IL, interleukin; imC, imidazole coordinated by carbon; imN, imidazole coordinated by nitrogen;ina, isonicotinic acid; isn, isonicotinamide; L-hist, L-histidine; LIT, liver infusion tryptose; LPS, lipopolysaccha-ride; NF-kb, nuclear factor-kb; NHE, normal hydrogen electrode; nic, nicotinamide; PBS, phosphate bufferedsaline; PCR, polymerase chain reaction; PE, phycoerythrin; py, pyridine; pz, pyrazine; SNP, sodium nitroprus-side; T. cruzi, Trypanosoma cruzi; TGF-b, transforming growth factor b; TNF-a, tumour necrosis factor-a

Introduction

Trypanosoma cruzi is an intracellular protozoan that is able tocause human disease and constitutes a major cause of heartdisease in Latin America. In Central and South America,Chagas’ disease affects 13 million people, with 75 million at

Correspondence: Paulo MM Guedes, Department of Biochemistry and Immu-nology, School of Medicine at Ribeirão Preto, Laboratory of Immunoparasitol-ogy, University of São Paulo, Avenue dos Bandeirantes 3900, Monte Alegre,14049-900 Ribeirão Preto, São Paulo, Brazil. E-mail: pauloguedes@usp.brReceived 10 April 2009; revised 22 September 2009; accepted 1 October 2009

British Journal of Pharmacology (2010), ••, ••–••© 2010 The AuthorsJournal compilation © 2010 The British Pharmacological Society All rights reserved 0007-1188/10www.brjpharmacol.org

risk for infection (WHO, 2005). Moreover, transmissionthrough transfusions and transplants represents a publichealth concern in non-endemic countries such as Australia,Canada, Spain and the United States (Schmunis, 2007). Thecurrent anti-parasitic chemotherapy with benznidazole (Bz)(Rochagan and Rodanil; Roche, Rio de Janeiro, Brazil) ornifurtimox (Lampit; Bayer, Wuppertal, Germany) is relativelyeffective during the acute and sub-chronic stages of Chagas’disease (Russomando et al., 1998; Sosa Estani et al., 1998;Cancado, 2002; Altclas et al., 2005). The adequacy of anti-parasitic treatment for patients in the indeterminate a ndchronic stages of the disease has not been confirmed. Somenon-randomised studies have suggested that trypanocidaltreatment with Bz slowed the progression of the clinical con-dition and cardiac function, as indicated by the prevention ofworsening ECG, of the patients (Viotti et al., 1994; 2006;Fabbro De Suasnabar et al., 2000; Lauria-Pires et al., 2000),while others yielded inconclusive results (Lauria-Pires et al.,2000). Unfortunately, Bz treatment is unsatisfactory duringthe chronic phase of the disease. Therefore, the developmentof new drugs is necessary.

Parasite elimination depends largely on the production ofpro-inflammatory cytokines, such as interferon-g (IFN-g),tumour necrosis factor-a (TNF-a) and interleukin (IL)-12, asthey act in concert to activate macrophages that kill intracel-lular parasites through the production of nitric oxide (NO)and its derived nitrogen and oxygen radicals (Aliberti et al.,1999; 2001; Machado et al., 2000). Studies using experimentalmodels of acute infection have demonstrated that the anti-parasitic activity of Bz involves the participation of thesecytokines (Michailowsky et al., 1998; Molina et al., 2000) aswell as covalent modifications of macromolecules by interme-diates of nitro-reducers (reductive stress) (Docampo, 1990).Conversely, nifurtimox acts by reducing the nitro group toform unstable nitro anion radicals, which, in turn, react toproduce highly toxic reduced oxygen metabolites, such assuperoxide anion and hydrogen peroxide (Docampo, 1990).

Both Bz and nifurtimox have significant side effects, includ-ing anorexia, vomiting, peripheral polyneuropathy and aller-gic dermopathy (Rassi et al., 1999). Moreover, several strainsof T. cruzi do not respond to these treatments, even during theacute phase of the disease (Filardi and Brener, 1987; Galvaoet al., 1993; Urbina, 1999). The rate of cure observed inpatients with these drugs is 50–70% during the acute phaseand 0–20% during the chronic phase (Guedes et al., 2006).This situation is severely aggravated by the absence of a goldstandard for diagnosis, which throws doubt on all the param-eters used for evaluating the outcome of trypanocidal thera-pies. Thus, there is an imperative requirement for thedevelopment of new therapeutic agents for Chagas’ disease.

Ruthenium NO donor compounds have recently emergedas an interesting and important alternative treatment ofexperimental infections with T. cruzi (Silva et al., 2007). Thesecompounds showed low toxicities in vitro and in vivo andstability in aqueous media in the presence of oxygen and NO,which are released by reducing agents present in the hostenvironment (Bogdan, 2001; Silva et al., 2007). Our grouprecently reported the trypanocidal activity, in vitro and in vivo,of a series of ruthenium nitrosyls, including trans-[RuII(NO+)(NH3)4L]X3, L = imidazole (imidazole coordinated by

nitrogen (imN) or imidazole coordinated by carbon, pyridine,L-histidine, sulphite (SO3

2-), pyrazine, nicotinamide, 4-picoline, triethylphosphite ([P(OEt)3]), isonicotinamide (isn),isonicotinic acid, X = BF4

-, Cl- or PF6-, and [RuII(NO+)(Hedta)]

against the Y strain of T. cruzi. Such compounds were efficientin reducing both parasitaemia and cardiac inflammation, andalso led to the total survival of infected mice that were treated(Silva et al., 2007). The therapeutic schedule, however, wasused only for 15 days of treatment and did not focus on theevaluation of parasitological cure. Here, we used a new andmore potent NO donor, trans-[RuCl([15]aneN4)NO]2+ complex([15]aneN4 = 1,4,8,12-tetraazacyclopentadecane, a macrocy-clic quadridentate amine ligand), over a therapeutic scheduleof 20 days of treatment in mice infected with the Y strain ofT. cruzi. The parasitological cure of mice treated with trans-[RuCl([15]aneN4)NO]2+ was evaluated and compared with thatafter treatment with Bz, as well as after a combination ofboth drugs.

Methods

Parasite and experimental infectionThe Y strain of T. cruzi was used in all experiments (Silva andNussenzweig, 1953). Mice were infected by i.p. injection with1 ¥ 103 blood trypomastigote stages of T. cruzi. Culture trypo-mastigotes were obtained from a fibroblast cell line (LLC-MK2). Epimastigote forms were obtained during theexponential phase of growth in liver infusion tryptose (LIT)medium.

Nitric oxide measurement in bone marrow-derived macrophagesBone marrow-derived macrophages (BMDM) were generatedfrom bone marrow stem cells that were cultured as previouslydescribed (Celada et al., 1984). These cells were then loadedwith the selective NO fluorescent dye 4,5-diaminofluoresceindiacetate (DAF-2 DA) (10 mM) for 30 min at room temperature(Rodrigues et al., 2008). The membrane permeable DAF-2 DAreadily enters the cells and is subsequently hydrolysed bycytosolic esterases to release free DAF-2, which does notleave the cell. At physiological pH, DAF-2 is relativelynon-fluorescent, but it forms the fluorescent product DAF-2triazole (DAF-2T) in the presence of NO and oxygen. Thisapproach allows the direct visualisation and semi-quantitative analysis of the basal NO availability at the cellu-lar level. The excess dye was removed by washing in a bathsolution. The cytosolic NO concentration was assessed by aLeica TCS SP5 spectral scanning confocal microscope (LeicaMicrosystems, Wetzlar, Germany). DAF-2T fluorescence wasexcited with the 488 hm line of an argon ion laser, and theemitted fluorescence was measured at 515 hm. Time coursesoftware was used to capture images of the cells at 2 s intervalsusing the Live Data Mode acquisition. The intensities of theintracellular maximum or minimum fluorescence were mea-sured through the application of the LSCM computer soft-ware. From these data, the basal fluorescence intensity value(basal) was registered at time zero of the experiment. The finalFI value (15-ane) was registered at 300 s after the addition of

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both trans-[RuCl([15]aneN4)NO]2+) (1 mM) and the reducingagent phenylephrine (0.1 mM) to the medium.

Culture of mouse leukaemic monocyte macrophage cell line264.7 macrophages and luciferase assaysThe murine mouse leukaemic monocyte macrophage cell line(RAW) 264.7 Luc macrophage, bearing the luciferase vectorinserted in the NF-kB promoter (pNF-kB-Luc), was routinelycultured in Dulbecco’s modified Eagle’s medium supple-mented with 10% fetal bovine serum (FBS) and penicillin-streptomycin, at 37°C in a humidified atmosphere of 5% CO2.For luciferase reporter assays, RAW 264.7 macrophages (5 ¥105 cells·mL-1) were grown in 24-well plates to 60–70% con-fluence. After culturing for 24 h, cells were stimulated withlipopolysaccharide (1 mg·mL-1) following the addition oftrans-[RuCl([15]aneN4)NO]2+ (1.0, 0.5 and 0.1 mM), ascorbicacid (AA; 1.0, 0.5 and 0.1 mM) and Bz (1.0 mM) to the growthmedium. Cells were treated at different drug concentrationsand kept overnight under incubation conditions. Cells wereharvested and extracted in lysis buffer (TNT), and theluciferase activities in the cell lysates were determined usingthe Dual Luciferase Reporter assay system (Promega). Datawere then expressed as a ratio of light units.

Surface molecule expression and cytotoxicity oftreated macrophageThe expression of co-stimulatory molecules (CD40, CD80 andCD86) and MHC-II was assessed in BMDM after being incu-bated for 24 h with trans-[RuCl([15]aneN4)NO]2+. Surfacestaining of untreated- and drug-treated macrophages was per-formed, in each case, by cell incubation with specificphycoerythrin- or fluorescein isothiocyanate-labelled mono-clonal antibodies: anti-CD40, anti-CD86, anti-CD86 andanti-MHC-II (BD-Pharmingen, San Diego, CA, USA). For cyto-toxicity assays, triplicates of BMDM were incubated withtrans-[RuCl([15]aneN4)NO]2+ (0.1, 0.5 and 1.0 mM) and/or AA(0.1, 0.5 and 1.0 mM) diluted in phosphate buffered saline(PBS) at 37°C in a humidified atmosphere of 5% CO2 for 24 h.Bz (1.0 mM) was used as the reference trypanocidal drug, andTween 20 (5.0, 0.5, and 0.005%) was used as a positive controlfor the death of cells. Cells were harvested and incubated withpropidium iodide at 50 mg·mL-1. Data were acquired after15 min, using a FACSorter apparatus (Becton-DickinsonImmunocytometry System Inc., San Jose, CA, USA). Multi-variate data analysis was performed in the FlowJo software(Ashland, OR, USA).

In vivo lethal dose of trans-[RuCl([15]aneN4)NO]2+

For the determination of the half-lethal dose (LD50) in vivo,three Swiss mice (30 g) per group were injected by i.p. injec-tion with trans-[RuCl([15]aneN4)NO]2+ or AA plus trans-[RuCl([15]aneN4)NO]2+ diluted in PBS at doses of 83, 250, 750,2000 and 6000 mmol·kg-1, respectively, with animal survivalobserved over the course of 48 h, as previously described(Silva et al., 2007).

Trypanocidal activity of trans-[RuCl([15]aneN4)NO]2+ in vitroThe in vitro anti-proliferative and trypanocidal activities oftrans-[RuCl([15]aneN4)NO]2+ were initially evaluated against

the epimastigote and trypomastigote forms, respectively, ofthe T. cruzi Y strains, as previously described (Silva et al.,2007). Trypomastigote and epimastigote cultures werere-suspended at a concentration of 6.5 106 parasites·mL-1 inRoswell Park Memorial Institute (RPMI) medium with 10%FBS and LIT, respectively. Triplicate cultures were treated withtrans-[RuCl([15]aneN4)NO]2+ (0.1, 0.5 and 1.0 mM) and/or AA(0.1, 0.5 and 1.0 mM) diluted in PBS at 37°C in a humidifiedatmosphere of 5% CO2. Bz (1.0 mM) (Roche) was used as thereference trypanocidal drug (positive control). Parasite viabil-ity was subsequently tested by determining the number ofmotile forms, according to the method established by Brener(1962b). The concentration of compound corresponding to50% anti-proliferative or trypanocidal activity after 24 h ofincubation was expressed as the IC50epi (inhibitory concentra-tion for the epimastigote form) or IC50try (inhibitory concen-tration for the trypomastigote form), respectively. The sameprotocol was used to determine the trypanocidal activity ofthe aqueous complex, trans-[RuCl([15]aneN4)H2O]+.

The intracellular action of the drugs was also determined onthe amastigote forms. Subconfluent monolayers of Vero cells(American Type Culture Collection CLL-81) were plated at1.25 ¥ 104 cells in RPMI (GIBCO, Grand Island, NY, USA)supplemented with 5% FBS (Nutricell, Campinas, São Paulo,Brazil) in each well of a eight-well chamber slide (chamberslide, Nunc Inc., Naperville, IL, USA) and incubated at 37°C ina humidified atmosphere of 5% CO2 for 24 h prior to infec-tion. Cell subconfluent monolayers were then infected withtissue culture trypomastigote forms (12.5 ¥ 104 parasites perwell) for 24 h. After infection, the chamber slides were washedtwice with cold PBS in order to remove parasites adhering tothe outside of cells, and the cells were then re-incubated inculture medium with drug tested. These experiments wereperformed in triplicate. The media was removed after 24 hincubation with drug and the chamber was stained withGiemsa, as previously described (Guedes et al., 2007). Thepercent of infected cells was determined by randomly exam-ining the slides and counting a minimum of 500 cells underthe microscope using high magnification (400¥).

Treatment of mice with NO donor and BzAll animal care and experimental protocols were approved bythe Ethics Committee on Animal Research of the Universityof São Paulo. The mice that were used in these experimentsbelonged to the animal stock of the Department of Biochem-istry and Immunology, Medicine School of Ribeirão Preto atthe University of São Paulo in São Paulo, Brazil. Animals werehoused in temperature-controlled rooms (22–25°C) andreceived water and food ad libitum in the animal facilities.Female Swiss mice 6–8 weeks old were infected with 1.0 ¥ 103

blood trypomastigotes per animal. A total of eight experimen-tal groups, each consisting of 10 Swiss mice that weighed25–30 g, were used. Treatment started soon after the detectionof parasitaemia, which occurred on day 4 post inoculation(p.i.). Bz, in a suspension in 4% gum arabica, was adminis-tered orally at 385 mmol·kg-1 of body weight per day(equivalent to 100 mg·kg-1) for 20 consecutive days. Trans-[RuCl([15]aneN4)NO]2+ (3.33 mmol·kg-1·day-1) and AA(3.33 mmol·kg-1·day-1) diluted in PBS were given by i.p.

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injection for 20 consecutive days. The groups received thefollowing treatments: Group 1 = PBS, Group 2 = AA, Group3 = Bz, Group 4 = trans-[RuCl([15]aneN4)NO]2+, Group 5 =trans-[RuCl([15]aneN4)NO]2+ plus Bz, Group 6 = trans-[RuCl([15]aneN4)NO]2+ plus AA, Group 7 = trans-[RuCl([15]aneN4)NO]2+ plus Bz plus AA and Group 8 = notinfected and not treated. Ten animals from each group werekilled at the end of the treatment (25 days p.i) for quantifi-cation of the levels of heart inflammation and cytokine pro-duction. Another 10 animals were used to evaluateparasitaemia, mortality and parasitological cure. The evalua-tion of parasitaemia was performed as previously described(Brener, 1962a).

Cytokine quantification by ELISAThe cytokine levels in serum samples obtained on day 25 p.i.were determined using ELISA kits for IL-10, IFN-g and TNF-a(all R&D Duoset, R&D, Minneapolis, MN, USA), according tothe manufacturers’ instructions. The reaction was detected byperoxidase-conjugated streptavidin followed by a substratemixture containing hydrogen peroxide and ABTS (SigmaAldrich, St. Louis, MO, USA) as a chromogen.

Nitrite measurement in the sera of treated animalsNitrite production in the sera of non-infected and infectedmice was measured by accumulation of nitrite, which is a NOmetabolite, in sera collected on day 25 p.i (the last day oftreatment), as described previously (Green et al., 1981).Briefly, 0.05 mL of Griess reagent [0.1% naphthyl ethylenedi-amine (Sigma Aldrich) and 1% sulfanilamide in 2.5% phos-phoric acid (Sigma Aldrich)] was added to 0.05 mL of sera,and absorbance was read at 540 nm using an automated platereader. Nitrite concentration was calculated from a NaNO2

standard curve.

Evaluation of parasitological cureParasitological cure was assessed by a battery of three inde-pendent tests, performed 30 days after the end of treatment,that is, 55 days p.i, including parasitological assays, such ashaemoculture and real-time PCR, and serological assays, suchas ELISA. The parasitological cure was determined basedupon a negative result with both the parasitological andserological methods.

The haemoculture assay was performed as follows. Animalswere bled from the orbital venous sinus, and 400 mL of bloodwere collected and divided into two tubes containing 3 mL ofLIT medium. Tubes were incubated at 28°C for 90 days andexamined monthly for the presence of parasites (Caldas et al.,2008).

Real-time PCR was performed using the Platinum® SYBR®Green qPCR SuperMix UDG with ROX system (Invitrogen,Carlsbad, CA, USA). The reaction mix included 2 mg of DNAplus 25 pmol of primers S35 (5′AAATAATGTACGGG (T/G)GAGATGCATGA3′) and S36 (5′GGGTTCGATTGGGGTTG-GTGT3′) (OPERON Technologies, INC). Samples were ampli-fied for 40 cycles in a 7000 Sequence Detection Systems(Applied Biosystems, Foster City, CA, USA), which generated aproduct of 330 bp.

ELISA and flow cytometry were performed with seraobtained on the first month after treatment. ELISA plates werecoated with T. cruzi antigen that was prepared from alkalineextraction of the T. cruzi Y strain during the exponentialphase of growth in LIT medium. Anti-mouse IgG-peroxidaseconjugated antibody (Sigma Aldrich) was used. The meanabsorbance for 10 negative control samples plus two standarddeviations were used as the cut-off to discriminate betweenpositive and negative results.

Quantification of myocardial inflammationMyocarditis was assessed as previously described (Guedeset al., 2007). Briefly, total nuclei numbers were determined in50 microscopic fields, using at least four representative, non-consecutive, haematoxylin and eosin-stained sections (thick-ness of 5 mm) from each mouse. Sections were examined witha Zeiss Integrationsplatte II eyepiece (Zeiss Co., Oberkochen,Germany) reticule used in conjunction with an Olympus BHSmicroscope (Olympus, Miami, FL, USA) at a final magnifica-tion of 400¥.

Statistical analysisData are expressed as the mean � SEM. Student’s t-test wasused to analyse the statistical significance of the differencesobserved between the infected and control assays. In the timecourse studies, two-way ANOVA was used, followed by Tukey–Kramer post hoc analysis. The Kaplan–Meier method was usedto compare survival curves of the studied groups. Differenceswere considered statistically significant when P < 0.05. Allanalyses were performed using the PRISM 3.0 software(GraphPad, San Diego, CA, USA)

MaterialsTrans-[RuCl([15]aneN4)NO]2+ was synthesised as described byBonaventura et al. (2004). The chemical structure of trans-[RuCl([15]aneN4)NO]2+ is shown in Figure 1. Bz (N-benzyl-2-

Figure 1 (A) Chemical structure of trans-[RuCl([15]aneN4)NO]2+;(B) 3Dd calculated, structure of trans-[RuCl([15]aneN4)NO]2+

(hydrogen atoms are omitted).

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nitro-1-imidazolacetamide) was from Roche, AA andphenylephrine from Sigma

Results

Trans-[RuCl([15]aneN4)NO]2+ has low toxicity and high LD50

Bz, which is the drug available for the clinical treatment ofChagas’ disease, is used at high doses and for long-term

therapy, with significant side effects. The toxicity of the newNO donor trans-[RuCl([15]aneN4)NO]2+ was determined andcompared with that of Bz using BMDM. AA, trans-[RuCl([15]aneN4)NO]2+ and Bz, all at doses of 1.0, 0.5 and0.1 mM, did not produce in vitro cytotoxicity (Figure 2). TheLD50 of trans-[RuCl([15]aneN4)NO]2+ for mice was approxi-mately 2000 mmol·kg-1, with the lethal dose ranging from2000 to 6000 mmol·kg-1.

Figure 2 Trans-[RuCl([15]aneN4)NO]2+ showed no in vitro toxicity in cultures of bone marrow macrophages. The in vitro toxicity assay ofbenznidazole (Bz; 1.0 mM) or the NO donor trans-[RuCl([15]aneN4)NO]2+ (NO donor, 0.1, 0.5 and 1.0 mM) alone, in combination withascorbic acid (AA; 0.1, 0.5 and 1.0 mM), or in combination with AA (0.1, 0.5 and 1.0 mM) plus Bz (1.0 mM) in bone marrow-derivedmacrophages (BMDM) after a 24-h incubation. Tween 20 (5.0, 0.5 and 0.005%) was used as positive control for cell death. (A) Percentmacrophage death at different concentrations and combinations of the compounds that were tested. (B) Representative FACS analysis plotsof BMDM after propidium iodide staining. Note the lack of damage on the BMDM target cells by trans-[RuCl([15]aneN4)NO]2+ Data representmeans � SEM and are representative of three independent experiments. SSC, side-scattered light; PBS, phosphate buffered saline. *Significantdifference (P < 0.05).

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Next, we tested whether the NO donor enhances the intra-cellular NO concentration, which affects the intracellularforms of T. cruzi. Macrophages that were cultured in the pres-ence of trans-[RuCl([15]aneN4)NO]2+ and 0.1 mM phenyleph-rine (reducing agent) exhibited enhanced intracellularconcentrations of NO (Figure 3). To verify if this effect wasdue to the direct activation of macrophages by trans-[RuCl([15]aneN4)NO]2+ or by its biologically reduced

by-products, BMDM were cultured in the presence of the NOdonor, and the expression levels of the co-stimulatory mol-ecules CD40, CD80, CD86 and MHC-II were analysed. Theaddition of drugs (NO donor, AA, and Bz) did not enhance theexpression of the co-stimulatory molecules in macrophages(Figure S1A). This absence of direct activation of macrophagesby the NO donor was confirmed by using murine RAWmacrophage-like cells that stably expressed a NF-kB luciferasereporter. After being cultured with the drugs, these reportercells did not show enhanced luciferase expression(Figure S1B). These results showed that the enhancement ofNO concentration in macrophages did not result from a gen-eralised activation of macrophages by the drugs, but rather asa result of the release of NO by trans-[RuCl([15]aneN4)NO]2+.

Trans-[RuCl([15]aneN4)NO]2+ has potent in vitrotrypanocidal activityAfter establishing the safety of trans-[RuCl([15]aneN4)NO]2+

through its low in vitro toxicity and wide therapeutic window,we evaluated its ability to inhibit all life cycle stages of T. cruzi.An assessment of the in vitro anti-proliferative activity againstepimastigotes revealed that Bz showed a 100% anti-proliferative effect at 1.0 mM. Treatment with the NO donoralone showed an approximately 10–20% anti-proliferativeactivity against the epimastigote form of T. cruzi. As theNO-releasing activity of the NO donor depends upon thepresence of reducing agents in the medium, treatment with acombination of the NO donor and AA showed 100% activityagainst the epimastigote form, similar to that of 1.0 mM Bz(Figure 4A). Interestingly, trans-[RuCl([15]aneN4)NO]2+ wasmore efficient at concentrations that were 100-fold lowerthan those of Bz against trypomastigotes, which are theT. cruzi forms present in the blood of vertebrate hosts(Figure 4B). At a concentration of 1.0 mM, Bz killed 53% oftrypomastigote forms after 24 h of incubation, whereas thetrans-[RuCl([15]aneN4)NO]2+ plus AA at 0.1, 0.5 and 1.0 mMkilled 100% of trypomastigote forms after 8 h (Figure 4B). ABz concentration of 10 mM was required to kill 100% ofparasites (data not shown). As observed against epimastigoteforms, the action of the NO donor against trypomastigoteforms also depended on the presence of reducing agents.

After NO release from trans-[RuCl([15]aneN4)NO]2+, the NOin the complex ion is replaced by H2O to form the complexion trans-[RuCl([15]aneN4)H2O+]. In order to determine if thision, devoid of NO, also has trypanocidal activity, the aqueouscomplex was incubated with the trypomastigote and epimas-tigote forms of T. cruzi. Throughout the range of concentra-tions that were used (0.1, 0.5 and 1.0 mM), approximately20% of the trypomastigotes were killed, and 30% of theepimastigotes displayed a reduction in proliferation(Figure 4C,D). The NO donor is thus likely to show a syner-gism between NO release and the nucleus of the metalliccompound.

Furthermore, in cultured Vero cells, trans-[RuCl([15]aneN4)NO]2+, in the presence of a reducing agent,was more efficient than Bz against the intracellular amastigoteform (Figure 4E). Vero cells were infected with T. cruzi andwere treated with drugs after parasite internalisation. The NOdonor did not show a significant effect against amastigote

Figure 3 Trans-[RuCl([15]aneN4)NO]2+ enhances cytosolic NOconcentration in macrophage cells from mice. Cultures of bonemarrow-derived macrophages (BMDM), treated or untreated withtrans-[RuCl([15]aneN4)NO]2+ (1.0 mM, for 100, 200 and 300 s) werepre-incubated in DAF-2DA (10 mM) for 30 min at room temperatureand examined under fluorescent light. (A) Significant enhancementof the DAF-2DA fluorescent green staining 300 s after NO donoraddition along with 0.1 mM phenylephrine (reducing agent), com-pared with the control culture (0.1 mM phenylephrine alone). Barsrepresent the mean � SEM of five different samples. *denotes statis-tical difference in the fluorescence intensity, P < 0.001. (B) Serialconfocal images from BMDM in culture medium (Control) and NOdonor-treated macrophages. Microphotographs representative offluorescence in macrophage cells were recorded before (mediumalone) and 100, 200 and 300 s after addition of the combination of1.0 mM of trans-[RuCl([15]aneN4)NO]2+ and 0.1 mM phenylephrine.

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Figure 4 Trans-[RuCl([15]aneN4)NO]2+ shows potent trypanocidal activity in vitro. The antiproliferative and trypanocidal activities (shown as%) of the NO donor trans-[RuCl([15]aneN4)NO]2+ (0.1, 0.5 and 1.0 mM) on epimastigote (A) and trypomastigote (B) forms of the Trypanosomacruzi Y strain in culture media. Parasites were treated with benznidazole (Bz, 1.0 mM) or NO donor alone, in combination with ascorbic acid(AA, 0.1, 0.5 and 1.0 mM), or in combination with AA plus Bz. The antiproliferative (C) and trypanocidal (D) activities of the aqueouscompound trans-[RuCl([15]aneN4)H2O]+ (0.1, 0.5 and 1.0 mM) were administered alone. Antiproliferative and trypanocidal activities oftrans-[RuCl([15]aneN4)NO]2+ and of the aqueous compound trans-[RuCl([15]aneN4)H2O]+ on epimastigote and trypomastigote forms of T.cruzi were determined by counting viable parasites at 8, 12,and 24 h post-treatment. The same concentrations described above fortrans-[RuCl([15]aneN4)NO]2+ were also used against amastigote forms (E). Cultures were stained by Giemsa dye. Each point is the mean � SEMand is representative of three independent experiments with similar results (n = 10). Statistically significant differences from control (P < 0.05)are denoted by *, #.

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forms of the parasite when it was added alone to theculture. After treatment with 1.0 mM of Bz or trans-[RuCl([15]aneN4)NO]2+ plus AA, the proportion of infectedcells was 14 � 4% and 2 � 0.5%, respectively, whereas, inuntreated cultures, there were 38 � 9% infected cells. Takentogether, these results indicate that the in vitro trypanocidaleffect of trans-[RuCl([15]aneN4)NO]2+ in the presence of areducing agent was greater than that of Bz.

The NO donor suppressed the parasitaemia, mortalityand myocarditisThe control untreated animals and those treated withAA alone showed similar parasitaemias, with the level ofparasitaemia peaking on the day 9 p.i. The parasitaemia wassuppressed between the second and fourth days of treatmentin all animals that were treated with either the NO donor(3.33 mmol·kg-1·day-1) alone or Bz (385 mmol·kg-1·day-1) alone.A combined treatment with both drugs suppressed the para-sitaemia on the first day of treatment. All control (untreated)animals showed significantly higher parasitaemia levels andpatent periods (P < 0.001) than animals treated with NOdonor and/or Bz (Figure 5A). In contrast to the in vitro effects,similar results were observed in mice treated with the NOdonor either alone or in combination with AA. This was dueto the presence of reducing agents in the blood and hosttissues. A survival rate of 100% of the T. cruzi- infected andtrans-[RuCl([15]aneN4)NO]2+ plus Bz-treated animals wasobserved for 6 months after the treatment, for all the thera-peutic schemes used. In contrast, untreated controls andanimals treated with AA alone showed a survival rate of 0 and10% respectively (Figure 5B).

Cardiac inflammation was evaluated at the end of the treat-ment (25 days p.i.). When administered separately, Bz and theNO donor were not able to achieve full clearance of thecardiac inflammation (Figure 5C,D), although these treat-ments showed significantly lower levels of myocardial inflam-mation (P < 0.05) than control animals treated with AA oruntreated animals. Interestingly, when administered together,Bz and trans-[RuCl([15]aneN4)NO]2+ eliminated myocarditis inmice (Figure 5C,D), whether or not AA was also added. AAwas used as reducing agent to assist NO release mainly in vitro.

Two months after each treatment, the animals were sepa-rated into cured or not cured groups, according to the resultsof the parasitological and serological tests. Both the cured andthe not cured animals that had been treated with trans-[RuCl([15]aneN4)NO]2+ and/or Bz showed no cardiac inflam-mation or significantly decreased cardiac inflammation,respectively, when compared with those animals that weretreated with AA (P < 0.05) (data not shown). The treated groupcould not be compared with the infected and untreatedanimals, because all the untreated mice died, as did thosetreated with AA, with only a single survivor in the latter groupat 1 month after treatment. The reduced myocarditis observedat the end of the treatment correlated with the poor produc-tion of NO and cytokines (IL-10, IFN-g and TNF-a) detected inthe sera of all treated animals (Figure S2A,B). As observed inthe cardiac inflammatory process 25 days p.i, nitrite andinflammatory cytokine production levels were high in theinfected and untreated animals and in the mice treated with

AA when compared with the animals treated with trans-[RuCl([15]aneN4)NO]2+ and/or Bz (Figure S2A,B). The parasi-tological cure was correlated with the absence of myocarditisand normalisation of serum cytokine and NO production inanimals that were treated with a combination of trans-[RuCl([15]aneN4)NO]2+ and Bz (Figure 5 and Figure S2A,B).

Parasitological cure was verified by three independent crite-ria, including haemoculture, real-time PCR and the presenceof specific IgG anti-T. cruzi antibodies detected by ELISA. Inmice treated with AA (group 1), Bz (group 2), trans-[RuCl([15]aneN4)NO]2+ (group 3), trans-[RuCl([15]aneN4)NO]2+

plus Bz (group 4), AA plus trans-[RuCl([15]aneN4)NO]2+ (group5) and AA plus trans-[RuCl([15]aneN4)NO]2+ plus Bz (group 6),the parasitological and serological tests were negative for 0, 40,30, 80, 20 and 80% of the animals respectively (Table 1).Serum levels of T. cruzi-specific immunoglobulin G antibodiesand parasite DNA levels in the animal groups treated with theNO donor plus Bz were absent or remarkably low, at levels thatwere very similar to those in the uninfected control group(Figure S3). Similar to the untreated control group, deathsoccurred among mice treated with AA during the acute phaseof the infection, with only one mouse surviving through tothe cure rate evaluation period.

Overall, these results indicate that treated but not curedanimals still had reductions in cardiac parasitism and incardiac damage, demonstrating the importance of treatment,even when the animals were not totally cured.

Discussion and conclusions

In this study, treatment with the new NO donor trans-[RuCl([15]aneN4)NO]2+ reduced parasitic load, cardiac inflam-mation, and allowed 100% survival in mice during the acutephase of an experimental infection with a highly virulentstrain of T. cruzi. In addition, this drug was able to induce aparasitological cure in 40% of the treated mice. Moreover,treatment in combination with Bz enhanced the rate of para-sitological cure up to 80%.

Currently available antiparasitic agents for the etiologicaltreatment of Chagas’ disease include Bz and nifurtimox. Theoutcomes obtained with both drugs are diverse, and their curerates depend on the stage of the disease as well as on thesensitivity of the parasite strain, which varies geographically(Filardi and Brener, 1987; Sosa Estani and Segura, 1999;Guedes et al., 2006). Despite being beneficial during the acutestage of infection, the outcomes of these antiparasitic thera-pies during chronic infection have not been systematicallyevaluated. Most Chagas’ disease cases are detected during thechronic phase of the disease, when cardiovascular and gas-trointestinal damage may be irreversible and when the onlyavailable therapeutic option may be surgery. Thus, the devel-opment of new therapeutic approaches is highly importantfor the treatment of Chagas’ disease. At the moment, a mul-ticentre, randomised double blind clinical trial is being con-ducted to test whether the administration of Bz to patientswith non-acute forms of the disease will prevent chroniccardiovascular damage (Marin-Neto et al., 2008).

NO plays multiple important roles in the immune response,including antiviral, antimicrobial, immunostimulatory,

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Figure 5 Treatment with trans-[RuCl([15]aneN4)NO]2+ suppresses parasitaemia, mortality and myocarditis of mice infected with T. cruzi. Swissmice were infected with 1 ¥ 103 blood trypomastigotes of T. cruzi Y strain, followed by different treatment schedules: ascorbic acid (AA;3.33 mmol·kg-1·day-1) alone, benznidazole (Bz; 385 mmol·kg-1·day-1) alone, NO donor trans-[RuCl([15]aneN4)NO]2+ (3.33 mmol·kg-1·day-1)alone, NO donor (3.33 mmol·kg-1·day-1) plus benznidazole (385 mmol·kg-1·day-1), NO donor (3.33 mmol·kg-1·day-1) in combination withascorbic AA (3.33 mmol·kg-1·day-1) and the NO donor (3.33 mmol·kg-1·day-1) in combination with AA (3.33 mmol·kg-1·day-1) and Bz(385 mmol·kg-1·day-1). Treatment was started on day 5 p.i and was given daily on 20 consecutive days. The control group received PBS.Parasitaemia (A) and survival (B) were evaluated daily over a period of 60 days post-infection. Representative sections of cardiac tissue (C) 25days after infection (the last day of treatment). Original magnification of microphotographs, ¥400. Quantification of cellular nuclei per 50 mm2

of heart tissue (D) of non-treated and treated animals. Note the synergic effect of the NO donor with Bz in eliminating myocarditis inT. cruzi-infected mice (Figure 5C,D), regardless of the presence or absence of AA. Each point is the mean � SEM and is representative of threeindependent experiments with similar results (n = 10). Statistically significant differences from control (P < 0.05) are denoted by *, #. Forexperimental details, refer to the legend in Table 1.

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immunosuppressive, cytotoxic and cytoprotective effects.The use of iNOS-/- mice demonstrated that most of theabove-described effects were mediated by iNOS-derived NO(Bogdan et al., 2000a,b; Nathan and Shiloh, 2000). NO,which is produced by activated macrophages, is crucial tothe intracellular killing of parasites, including T. cruzi(Martins et al., 2001). Many protozoa are able to inhibit NOproduction, which thus allows them to evade the immunesystem. T. cruzi induces apoptosis in lymphocytes, and regu-lation of iNOS mediated by cell–cell contact has been seenin apoptotic lymphocytes (Freire-de-Lima et al., 2000).Uptake of apoptotic cells drives the growth of T. cruzi withinmacrophages. The uptake of apoptotic, but not necrotic,lymphocytes by macrophages through fibronectin receptorsand CD36 leads to the down-regulated expression of iNOSand, thus, shifts L-arginine metabolism towards the arginasepathway. In turn, there is increased production of ornithineand putrescine, which are known growth factors for T. cruzi.These effects result from the induction of endogenous TGF-band immune system evasion (Freire-de-Lima et al., 2000). AsNO produced within phagocytes is crucial to parasite killing,therapeutic approaches that use NO-releasing compoundscould improve the protective immune response and are thuspromising agents for the treatment of this intracellularpathogen.

In fact, the treatment of T. cruzi-infected, immunosup-pressed mice with Bz generates lower survival and cure ratesthan the same treatment in immunocompetent animals,which indicates that the protective effects of Bz, particularlythe processes of macrophage activation, NO production andparasite destruction, are dependent on the integrity of theimmune system (Silva et al., 1995; Molina et al., 2000).Recently, our group demonstrated that NO donors adminis-tered at low concentrations are capable of killing T. cruzi (Silvaet al., 2007). The rate of parasitological cure for these com-pounds has not been determined. Moreover, the NO donorused in the present study showed better effects against T. cruzithan those previously tested by our group, which is likely dueto the longer period of NO release (Bonaventura et al., 2004)

and synergistic activity with the aqueous ion formed fromtrans-[RuCl([15]aneN4)NO]2+.

We demonstrated that trans-[RuCl([15]aneN4)NO]2+ did notshow in vitro toxicity to bone marrow macrophages. In addi-tion, the cytosolic NO concentration within macrophages inculture was remarkably enhanced after the addition of trans-[RuCl([15]aneN4)NO]2+ along with the reducing agent phe-nylephrine to the culture medium. The therapeutic dose usedin vivo was well below its LD50 (3.33 mmol·kg-1 vs. 2000–6000 mmol·kg-1). Even when administered at lower concentra-tions, trans-[RuCl([15]aneN4)NO]2+ was more efficient than Bzin killing the epimastigote, trypomastigote and amastigoteforms of T. cruzi. Although trans-[RuCl([15]aneN4)NO]2+ wasvery effective in vitro, it only generated a 30% parasitologicalcure rate in vivo when administered alone. The cure rate of adrug correlates with its pharmacokinetic characteristics, suchas bioavailability, frequency, pathway of administration andother factors. This particular NO donor drug increases thetime over which NO is released, which may have an effect onits cure rate. Evaluation of these factors in the murine modelwill constitute the next steps in our research. As theNO-releasing activity of trans-[RuCl([15]aneN4)NO]2+ lasts for2 h (Bonaventura et al., 2004), a schedule of giving trans-[RuCl([15]aneN4)NO]2+ two or three times a day would prob-ably improve its pharmacodynamic properties. Based on thefindings reported in this paper, future studies can be con-ducted to determine if and how the NO release period can beextended.

We observed 100% of survival in mice treated with3.33 mmol·kg-1·day-1 of trans-[RuCl([15]aneN4)NO]2+, eitheralone or in combination with 385 mmol·kg-1·day-1 of Bz, whileall the infected and untreated animals died. The NO donorstested against T. cruzi, however, are not bioequivalent, sincethe survival rates of mice treated with Ru(NO)imN orRu(NO)isn at the same molar dose were 40 and 60% respec-tively (Silva et al., 2007). These results demonstrate that thetrans-[RuCl([15]aneN4)NO]2+ and other NO donors that hadbeen previously tested are more potent and safer than Bz. Ourresults are promising, given that the NO donor trans-

Table 1 The NO donor trans-[RuCl([15]aneN4)NO]2+ produced parasitological cure of experimental Chagas’ disease

Therapeutic schedulea Positive results/testb No of positive testspost-treatment/total (%)

Cure rate (%)

No of positiveELISA/total

No of positivehaemoculture/total

No of positive real-timePCR/total

Ascorbic acidc (AA) 1 1 1 1/1 (100) 0Benznidazole (Bz) 6 5 6 6/10 (60) 40NO donor 7 6 7 7/10 (70) 30NO donor + Bz 2 1 2 2/10 (20) 80NO donor + AA 8 4 8 8/10 (80) 20NO donor + AA + Bz 2 2 2 2/10 (20) 80

aThe effects of the NO donor, trans-[RuCl([15]aneN4)NO]2+, given alone and/or: AA, Bz as well as the NO donor plus ascorbic acid plus benznidazole on miceinfected with T. cruzi Y strain on measures of cure are shown.Swiss female mice, 28–30 g, were infected with 1 ¥ 103 blood trypomastigotes of T. cruzi Y strain. Treatment started after detection of patent parasitaemia, on day5 p.i. given daily over a period of 20 days. The NO donor trans-[RuCl([15]aneN4)NO]2+, and AA were injected i.p. at 3.33 mmol·kg-1·day-1. Bz was administeredorally at 385 mmol·kg-1. The infected control group received PBS. Data are expressed as the mean � SEM and are representative of three independent experimentswith similar results, n = 10. PBS, phosphate buffered saline.bResults shown are the number of positive ELISA, haemoculture and real time PCR tests. Ten mice were used for each treatment group and for the not infectedand T. cruzi-infected controls.cOnly one animal remained alive until the period of the cure rate evaluation in the control group. No survivals were observed in the PBS, untreated control group.

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[RuCl([15]aneN4)NO]2+ displays characteristics that complywith the essential criteria for antiparasitic drugs specified bythe WHO and other organisations running control pro-grammes in affected countries.

The cardiac inflammatory process was decreased at the endof the treatment with trans-[RuCl([15]aneN4)NO]2+, with Bz orwith their combination (26 d.p.i.). A less intense reduction ofmyocarditis is observed with other NO donors and Bz(Andrade et al., 1991; Segura et al., 1994; Silva et al., 2007).However, the combination of NO donor and Bz resulted inthe absence of myocarditis by the end of the treatment. More-over, animals that were treated but not considered to be curedshowed a reduced level of cardiac inflammation when com-pared with the control (animals treated with AA alone). Theseanimals also showed decreased parasitism. As the presence ofparasites is believed to play an important role in causingcardiac lesions, specific drug-combination therapies mightreduce the myocardial damage and morbidity during theacute and chronic phases of the disease in human beings(Coura et al., 1997). Data from the literature also indicate thereversibility of cardiac fibrosis in mice chronically infectedwith T. cruzi and undergoing specific chemotherapy (Andradeet al., 1991).

New drugs for treating Chagas’ disease should be able toimprove the quality of life for those infected, through a reduc-tion of the chronic morbidity, the subsequent expenditure bygovernment health systems and early retirement in develop-ing countries. In conclusion, our results indicate thattrans-[RuCl([15]aneN4)NO]2+ has both in vitro and in vivo try-panocidal activity and is capable of inducing a radical para-sitological cure in a mouse model of acute infection.Furthermore, this compound can be used at high doses andrepresents a potentially useful candidate for the treatment ofChagas’ disease in human beings.

Acknowledgements

We thank Cristiane Maria Milanezi for her excellent technicalassistance. This project received support from Fundação deAmparo a Pesquisa do Estado de São Paulo (FAPESP 2007/53940-0), Conselho Nacional de Desenvolvimento Científicoe Tecnológico (CNPq 410467/2006-5) and a Research fellow-ship from Coordenação de Aperfeiçoamento de Pesssoal deNível Superior (CAPES).

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Supporting information

Additional Supporting Information may be found in theonline version of this article:

Figure S1 Trans-[RuCl([15]aneN4)NO]2+ did not enhance theexpression of co-stimulatory molecules and did not activatemacrophages. In vitro effect of different drug schedules on theexpression of CD40, CD80 and CD86 co-stimulatory andMHC-II molecules in blood bone marrow-derived macroph-ages.1 Drug treatments corresponding to each set of barsstanding for a co-stimulatory or MHC-II molecule in the his-togram: medium – no drug, 1 mg/mL LPS, 1.0 mM trans-[RuCl([15]aneN4)NO]2+ plus 1.0 mM ascorbic acid, 0.5 mMascorbic acid, 0.5 mM trans-[RuCl([15]aneN4)NO]2+, 1.0 mMtrans-[RuCl([15]aneN4)NO]2+ plus 1.0 mM benznidazole,0.5 mM trans-[RuCl([15]aneN4)NO]2+ plus 0.5 mM ascorbicacid plus 0.5 mM benznidazole. LPS was used as a positivecontrol for macrophage activation. Macrophage cultures weremaintained for 24 h under incubation conditions followingthe expression analysis of the co-stimulatory and MHC-IImolecules by flow cytometry (A). The murine macrophage-like cell line RAW 264.7 Luc, bearing the luciferase vectorinserted in the NF-kB promoter, was treated with the sametherapeutic schedule as above under incubation conditionsfor 24 h, prior to the assessment of luciferase activity (B). Data(means � standard error of the mean) are representative ofthree independent experiments with similar results (n = 10).Statistically significant differences from control (unpairedt-test, P < 0.05) are denoted by *.Figure S2 Treatment with trans-[RuCl([15]aneN4)NO]2+

reduced cytokine and NO production at the end of the thera-peutic schedule. Swiss mice were inoculated with 1 ¥ 103

blood trypomastigotes of T. cruzi Y strain and treated withtrans-[RuCl([15]aneN4)NO]2+ (3.33 mmol/kg/day), and/orascorbic acid (3.33 mmol/kg/day), and/or benznidazole (385mmol/kg/day) for 20 consecutive days. The control groupreceived only PBS. For details on the experimental protocol,refer to the legend in Figure 5. Nitrite (A) and IL-10, IFN-g andTNF-a (B) production were determined in the blood sera onday 25 post-infection (the last day of treatment) by ELISA andthe Griess method, respectively. Data are representative ofthree independent experiments with similar results, n = 10.Data represent means � SEM. *, + and # P < 0.05, differentsymbols indicate significant difference.

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Figure S3 The treatment with trans-[RuCl([15]aneN4)NO]2+

induces a parasitological cure in T. cruzi-infected mice. Swissmice were inoculated with 1 ¥ 103 blood trypomastigotes of T.cruzi Y strain and treated with trans-[RuCl([15]aneN4)NO]2+

(3.33 mmol/kg/day), and/or ascorbic acid (3.33 mmol/kg/day), and/or benznidazole (385 mmol/kg/day) for 20 consecu-tive days. The control group received only PBS. For details onthe experimental protocol, refer to the legend in Figure 5. Thequantification of T. cruzi-specific immunoglobulin G antibod-ies in the blood sera (A) and real-time PCR analysis for thepresence of T. cruzi DNA in the myocardium (B) were per-formed 55 days after infection (1 month after the end oftreatment). Note that the serum levels of T. cruzi-specific

immunoglobulin G antibodies as well as the levels of parasiteDNA in the animal groups treated with the NO donor plus Bzare remarkably low and are very similar to what is seen for theuninfected control group. Data represent means � SEM andare representative of three independent experiments withsimilar results (unpaired t-test, P < 0.05) (n = 10). PBS, phos-phate buffered saline.

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NO donor therapy for Chagas’ diseasePMM Guedes et al 13

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